CN112546099A

CN112546099A - Buckwheat seedling leaching liquor, application of buckwheat seedling leaching liquor in preparation of cell antioxidant improving composition and/or liver health-care composition and preparation method

Info

Publication number: CN112546099A
Application number: CN202010949833.8A
Authority: CN
Inventors: 林咏翔; 姚采涵
Original assignee: Bayote Biotechnology Shanghai Co ltd
Current assignee: Bayote Biotechnology Shanghai Co ltd
Priority date: 2019-09-10
Filing date: 2020-09-10
Publication date: 2021-03-26
Also published as: TW202110466A; CN112546058A; CN112546035A; TWI794575B; TWI749435B; TW202112361A; TW202112381A; TWI774055B

Abstract

Use of buckwheat sprout extract for preparing a composition. The composition has one or more of the following functions: improving the antioxidant activity of cells, improving the metabolic activity, and improving the synthesis of Glutathione (GSH) in cells.

Description

Buckwheat seedling leaching liquor, application of buckwheat seedling leaching liquor in preparation of cell antioxidant improving composition and/or liver health-care composition and preparation method

Technical Field

The invention relates to the use of buckwheat sprout leach liquor, in particular to the use of buckwheat sprout for preparing a composition for improving cell antioxidation and/or liver health care.

Background

Since the development of organic and natural dietary concepts, biotechnology companies and food manufacturers have actively invested in the development of products related to natural plants. In order to enable plant-related products to have a scientific verification basis for body health help, the analysis of active ingredients and the evaluation of efficacy of plants become key projects for product development.

Among many natural plants, buckwheat can grow in poor acid soil without requiring excessive nutrients. Buckwheat is commonly used, for example, in the production of buckwheat flour, in brewing, or as tea beverages. If other efficacies of buckwheat can be developed, buckwheat can be further effectively utilized, and the buckwheat is more beneficial to the health and welfare of the whole people.

Disclosure of Invention

In order to achieve the above objects, the present invention provides a buckwheat sprout leaching liquor, its use for preparing a composition for improving cell antioxidation and/or a composition for liver health and a preparation method thereof.

In one embodiment, the use of a buckwheat seedling extract for preparing a composition for increasing antioxidant activity of cells.

In some embodiments, the use of a buckwheat sprout extract is for preparing a composition for liver health.

In some embodiments, the buckwheat sprout extract is used to increase the antioxidant and metabolic activity of the cells.

In some embodiments, the buckwheat sprout extract is used to enhance the synthesis of Glutathione (GSH) in cells.

In some embodiments, the buckwheat sprout extract is obtained by extracting buckwheat sprouts with a solvent, and the buckwheat sprouts are seedlings that grow for 6 to 12 days after the buckwheat seeds germinate.

In some embodiments, the solvent is water, the weight ratio of the solvent to the buckwheat seedlings is 2-5:1, the extraction temperature of the buckwheat seedlings is between 80 ℃ and 90 ℃, and the extraction time of the buckwheat seedlings is 120 minutes.

In some embodiments, the buckwheat sprout extract is 4.7 ° Bx to 5.3 ° Bx.

In some embodiments, the buckwheat sprout extract has a flavone content of about 815 μ g/mL.

In some embodiments, the buckwheat sprout leachate comprises at least one of the following bioactive substances: guanosine (Guanosine), Vitexin (Vitexin), Isovitexin (Isovitexin), Rutin (Rutin), isoorientaxin (Isoorientin), and Luteolin 8-C-glucoside (Luteolin-8-C-glucoside).

In some embodiments, guanosine is used to enhance glutathione (glutathione) synthesis in cells.

In some embodiments, a method for preparing buckwheat sprout leachate comprises: leaching buckwheat seedlings with water in a weight ratio of 2-5:1 at a temperature of 80-90 ℃ for 60-180 minutes to obtain a leaching stock solution; and filtering the raw solution to obtain a buckwheat seedling leaching solution.

In some embodiments, the buckwheat seedlings used for extraction are seedlings that grow 6 to 12 days after the buckwheat seeds germinate.

In some embodiments, the method for preparing buckwheat sprout leachate further comprises: concentrating the buckwheat sprout leaching solution under reduced pressure until Brix value (Degrees Brix) of the buckwheat sprout leaching solution is 4.7-5.3 (i.e. 4.7 ° Bx-5.3 ° Bx) to obtain concentrated buckwheat sprout leaching solution.

In some embodiments, the aforementioned reduced pressure concentration step is performed at 50 ℃ to 60 ℃.

In some embodiments, the concentrated buckwheat sprout extract has a total flavone content of 815 μ g/mL.

In some embodiments, the buckwheat sprout leachate obtained after filtration comprises at least one of the following bioactive substances: guanosine (Guanosine), Vitexin (Vitexin), Isovitexin (Isovitexin), Rutin (Rutin), isoorientaxin (Isoorientin), and Luteolin 8-C-glucoside (Luteolin-8-C-glucoside).

The invention has the beneficial effects that: in any embodiment, the use of buckwheat sprout leachate in the preparation of a composition for enhancing cellular antioxidant and/or liver health and a method for preparing buckwheat sprout leachate suitable for providing buckwheat sprout leachate or a composition thereof. Wherein the buckwheat sprout leaching liquor or the composition thereof has one or more of the following functions: improving the antioxidant activity of cells, improving the metabolic activity, and improving the synthesis of Glutathione (GSH) in cells.

Drawings

FIG. 1 is HPLC fingerprint of buckwheat sprout leaching solution obtained from buckwheat sprout growing for 0, 3, 6, 9, 12, 15, 18 days after the buckwheat seed germinates.

Fig. 2 is a hydrogen spectrum of bioactive substance 01.

Fig. 3 is a hydrogen spectrum of the bioactive substance 02.

Fig. 4 is a hydrogen spectrum of the bioactive substance 03.

Fig. 5 is a hydrogen spectrum of bioactive 04.

Fig. 6 is a hydrogen spectrum of bioactive 05.

Fig. 7 is a hydrogen spectrum of the bioactive substance 06.

FIG. 8 is a graph showing the results of experiments on the relative amounts of ROS produced in the control group, the experimental group 1, and the experimental group 2.

Fig. 9 is a graph showing the results of experiments on the relative production amounts of glutathione (glutathione) in the control group, the experimental group 1, and the experimental group 2.

FIG. 10 is a graph showing the experimental results of the reducing power of the buckwheat seedling leaching solution.

Fig. 11 is a graph showing the results of the control, experiment 1, experiment 2, experiment 3, experiment 4, experiment 5, and experiment 6 on the relative production of glutathione (glutathione).

Reference numerals

Time points A, B, C, D, E, F, G

Detailed Description

The invention will be described in detail with reference to the following drawings, which are provided for illustration purposes and the like:

as used herein, the concentration symbol "wt%" generally refers to weight percent concentration, while the concentration symbol "vol%" generally refers to volume percent concentration. As used herein, the term "leaching" refers to a process of leaching some components of a plant by immersing the plant (in a solid state) in a solvent (in a liquid state), and the term "leaching" refers to a juice produced by immersing the plant (in a solid state) in a solvent (in a liquid state) to leach some components of the plant. It should be understood that the term "leaching" as referred to herein may be used interchangeably with the term "extracting" and that the term "biologically active substance" as referred to herein may be used interchangeably with the term "compound".

In one embodiment, the use of a buckwheat sprout extract for preparing a composition for increasing antioxidant activity of cells.

In one embodiment, the use of buckwheat sprout extract is used for preparing a composition for liver health care.

In some embodiments, buckwheat sprout extracts are used to enhance the synthesis of Glutathione (GSH) in cells.

In some embodiments, the buckwheat sprout leach liquor comprises at least one of the following bioactive substances: guanosine (Guanosine), Vitexin (Vitexin), Isovitexin (Isovitexin), Rutin (Rutin), isoorientaxin (Isoorientin), and Luteolin 8-C-glucoside (Luteolin-8-C-glucoside).

Among them, guanosine can be used to enhance the synthesis of glutathione (glutathione) in cells.

In some embodiments, guanosine is of the formula 1.

In some embodiments, vitexin has the formula 2 below.

In some embodiments, the isovitexin has the formula 3 below.

In some embodiments, the rutin is of the formula 4 below.

In some embodiments, the isoorientasin has the structural formula 5 below.

In some embodiments, the luteolin 8-C-glucoside has the structural formula 6 below.

In some embodiments, the buckwheat sprout extract can be obtained from young seedlings of buckwheat (hereinafter referred to as buckwheat sprouts). The preparation method of the buckwheat seedling leaching liquor comprises the following steps: leaching buckwheat seedlings with water in a weight ratio of 2-5:1 at a temperature of 80-90 ℃ for 60-120 minutes to obtain a leaching stock solution; and filtering the raw solution to obtain a buckwheat sprout leaching solution (filtrate).

In some embodiments of the leaching step, the buckwheat seedlings are seedlings that grow 6 to 12 days after the buckwheat seeds germinate. In some embodiments of the leaching step, the buckwheat seedlings may preferably be seedlings that grow for 9 days after the buckwheat seeds germinate. In some embodiments of the leaching step, the buckwheat sprouts may be tartary buckwheat (Fagopyrum tataricum). In some embodiments of the leaching step, the buckwheat seedling may include a cotyledon of the seedling, an embryonic axis of the seedling, and a root of the seedling. In some embodiments of the leaching step, the weight ratio of water to buckwheat sprouts may be 3: 1.

In some embodiments, the filtering step may be performed by filtering the raw leaching solution obtained in the previous step with a 400 mesh sieve to remove fine solids.

In some embodiments, the method for preparing the buckwheat sprout leaching solution may further comprise: concentrating the buckwheat sprout leaching solution under reduced pressure until Brix value (Degrees Brix, Bx) of the buckwheat sprout leaching solution is 4.7-5.3 to obtain concentrated buckwheat sprout leaching solution (i.e. concentrated solution). Wherein the concentration under reduced pressure step can be carried out at 50 ℃ to 60 ℃.

In some embodiments, the total flavone content of the concentrated buckwheat sprout leachate can be 815 μ g/mL.

In some embodiments, the buckwheat sprout leaching solution can be the filtrate obtained from the filtration step or the concentrated solution obtained from the reduced pressure concentration step. In some embodiments, the composition may be a pharmaceutical. Changing , the medicine contains buckwheat sprout leachate with effective content.

In some embodiments, the aforementioned medicaments may be formulated into dosage forms suitable for enteral, parenteral (parenterally), oral, or topical (topically) administration using techniques well known to those skilled in the art.

In some embodiments, the dosage form for enteral or oral administration may be, but is not limited to, tablets (tablets), troches (troche), buccal tablets (dosage), pills (pill), capsules (capsule), dispersible powders (dispersible granules) or granules (granules), solutions, suspensions (suspensions), emulsions (emulsions), syrups (syrup), elixirs (elixir), syrups (syrup), or the like. In some embodiments, parenteral or topical administration forms can be, but are not limited to, injections (injections), sterile powders (sterile powders), external preparations (external preparations), or the like. In some embodiments, the administration of the injectate can be subcutaneous (subcutaneous), intradermal (intraepithelial injection), or intralesional (intrafocal injection).

In some embodiments, the aforementioned pharmaceutical may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) widely used in pharmaceutical manufacturing technology. In some embodiments, the pharmaceutically acceptable carrier can be one or more of the following carriers: solvents (solvents), buffers (buffers), emulsifiers (emulsifiers), suspending agents (suspending agents), disintegrating agents (disintegrants), disintegrating agents (disintegrating agents), dispersing agents (dispersing agents), binding agents (binding agents), excipients, stabilizing agents (stabilizing agents), chelating agents (chelating agents), diluents (diluents), gelling agents (gelling agents), preservatives (preserving), wetting agents (wetting agents), lubricants (lubricants), absorption delaying agents (absorption delaying agents), liposomes (liposomes), and the like. The type and amount of carrier selected for use is within the skill of one of ordinary skill in the art. In some embodiments, the solvent as a pharmaceutically acceptable carrier may be water, normal saline (normal saline), Phosphate Buffered Saline (PBS), or an aqueous solution containing alcohol (alcohol stabilizing aqueous solution).

In some embodiments, the aforementioned composition may be an edible composition. In other words , the edible composition contained buckwheat sprout extracts in specified amounts. In some embodiments, the aforementioned edible composition may be a food product or food additive (food additive). In some embodiments, the food product may be, but is not limited to: beverages (leafages), fermented foods (fermented foods), bakery products (bakery products), health foods (health foods) or dietary supplements (dietary supplements).

In some embodiments, the aforementioned edible composition can be administered orally to a subject. The edible composition can be in the form of powder, granule, solution, colloid or paste.

In some embodiments, the aforementioned composition may be a cosmetic or a care product. In other words , the cosmetic or health product contains buckwheat sprout extracts in specified amounts.

In some embodiments, the cosmetic or care product may be any of the following types: lotions, gels, jellies, mud masks, lotions, creams, lipsticks, foundations, pressed powders, honey powders, make-up removers, facial cleansers, shower gels, shampoos, hair tonics, sun blocks, hand creams, nail polishes, perfumes, essences, and facial masks. In some embodiments, the cosmetic or care product may further comprise an external acceptable ingredient, if desired. In some embodiments, the topical acceptable ingredient can be, for example, an emulsifier, a penetration enhancer, a softener, a solvent, an excipient, an antioxidant, or a combination thereof.

The first embodiment is as follows: preparation of buckwheat seedling leaching liquor

The experimental steps are as follows:

1. obtaining un-germinated seeds of radix Et rhizoma Fagopyri Tatarici (growth for 0 day) and seedlings of radix Et rhizoma Fagopyri Tatarici (growth for 3 days, 6 days, 9 days, 12 days, 15 days and 18 days after germination), and collecting seven groups of raw materials.

2. The raw material obtained in the previous step was leached with water at a temperature of 85 ℃ for 120 minutes to obtain each group of solids-containing leach stock. Wherein the weight ratio of the water to the raw materials is 3: 1.

3. And filtering the seven groups of the leaching stock solution obtained in the previous step by using a filter screen with 400 meshes to remove fine solids to obtain the buckwheat seedling leaching liquor of each group.

4. Concentrating the seven groups of buckwheat sprout leaching liquor obtained in the previous step at 55 deg.C under reduced pressure by a concentrator (BUCHI-Rotavapor R-100) until Brix value (Degrees Brix, DEG Bx) of buckwheat sprout leaching liquor is 5 to obtain concentrated buckwheat sprout leaching liquor.

Hereinafter, a concentrated buckwheat seedling leaching solution obtained by using a seed which has not yet germinated as a raw material is referred to as a leaching solution 1, a concentrated buckwheat seedling leaching solution obtained by using a buckwheat seedling which has grown for 3 days as a raw material is referred to as a leaching solution 2, a concentrated buckwheat seedling leaching solution obtained by using a buckwheat seedling which has grown for 6 days as a raw material is referred to as a leaching solution 3, a concentrated buckwheat seedling leaching solution obtained by using a buckwheat seedling which has grown for 9 days as a raw material is referred to as a leaching solution 4, a concentrated buckwheat seedling leaching solution obtained by using a buckwheat seedling which has grown for 12 days as a raw material is referred to as a leaching solution 5, a concentrated buckwheat seedling leaching solution obtained by using a buckwheat seedling which has grown for 15 days as a raw material is referred to as a leaching solution 6, and a concentrated buckwheat seedling leaching solution obtained by using a buckwheat seedling which has grown for 18 days.

Example II, component analysis of buckwheat seedling extract

Taking the leaching solution 1 to the leaching solution 7 obtained in the first step, and analyzing the fingerprint of the leaching solution 1 to the leaching solution 7 by using a Liquid chromatography-mass spectrometry (LC-MS) technology.

Firstly, fingerprint spectrum analysis.

The experimental steps are as follows:

1. and (3) concentrating the leaching solution 1 to the leaching solution 7 obtained in the first step to remove the solvent to obtain solids 1 to 7. Solids 1 to 7 were prepared as 10 milligrams per milliliter (mg/mL) of sample 1 to sample 7, respectively, with water as the solvent, for a total of 7 samples.

2. 10 μ L of each sample from the previous step was analyzed by High Performance Liquid Chromatography (HPLC) (using a Mightysil RP-18GP 250, 250X 10mm,5 μm column from Kanto chemical Co., Japan). Wherein, the pump system of the high performance liquid chromatography is Hitachi L-2310series, the detector model is Hitachi L-2420UV-VIS, and the data processing software is D-2000 Elite.

In this step, the flow rate of the mobile phase was controlled to 1mL/min, and the detection wavelength was set to 280nm, and gradient elution was performed. The gradient change of the mobile phase is shown in the following table one: mobile phase a was 0.1 vol% formic acid (in water) and mobile phase B was 0.1 vol% formic acid (in methanol).

Watch 1

Extraction time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	98	2
10	98	2
			40	30	70
50	0	100
			60	0	100
62	98	2
			70	98	2

The experimental results are as follows:

the HPLC fingerprints for leach 1 to leach 7 are shown in figure 1. Referring to fig. 1, there are distinct peaks in the fingerprints of leachate 3 to leachate 5 at time point A, B, C, D, E, F and at the position of G. The area of each peak at time point A, B, C, D, E, F and G in FIG. 1 was calculated by data processing software integration calculations. Then, based on the obtained peak areas, the ratio of the maximum peak area to the minimum peak area is calculated, and the calculation results of each sample are shown in the following table two. Referring to fig. 1 and table two, the growth metabolites (hereinafter referred to as metabolites) separated by leachate 3 through leachate 5 at time point A, B, C, D, E, F and G were significantly higher than the other leachate (i.e., there was a significant peak). The content of the metabolite separated at time point A, D, F and G in the leaching solution 4 (i.e., the leaching solution of buckwheat seedlings obtained by using buckwheat seedlings growing for 9 days after buckwheat seeds germinate) was the highest value (i.e., the peak value of the peak value was the highest) compared to the leaching solution of buckwheat seedlings obtained by using buckwheat seedlings growing for other days after buckwheat seeds germinate. The content of the metabolite separated from the leaching solution 4 at the time point B, C, E is less than that of the leaching solution 5 (buckwheat sprout extract obtained by using buckwheat sprouts growing for 12 days after the germination of buckwheat seeds as raw materials). Therefore, it was found that the buckwheat sprout leachate obtained from the buckwheat sprouts growing for 6 to 12 days after the buckwheat seeds germinated had a high content of metabolites, and that the buckwheat sprout leachate obtained from the buckwheat sprouts growing for 9 days after the buckwheat seeds germinated had a higher content of metabolites than the buckwheat sprout leachate obtained from the buckwheat sprouts growing for other days after the buckwheat seeds germinated.

Watch two

	A	E	G	F	D	C	B
								Leach liquor 1	1.01	1.00	1.00	-	-	-	-
Leach liquor 2	1.95	1.80	1.89	1.38	1.37	1.39	1.46
								Leach liquor 3	3.20	6.33	8.82	16.47	10.98	15.35	46.49
Leach liquor 4	3.25	8.04	11.99	23.54	15.27	21.47	54.69
								Leach liquor 5	2.63	8.78	12.69	21.46	15.74	20.29	21.21
Leach liquor 6	1.29	1.43	1.49	1.20	1.14	1.16	1.31
								Extract 7	1.00	1.26	1.28	1.00	1.00	1.00	1.00

(II) analysis of metabolites

The collected separated liquids at time point A, C, D, E, F and G in the above experiment (I) were analyzed by Nuclear Magnetic Resonance (NMR) spectrometer (type: Ascend 400MHz, Bruker Co) to obtain hydrogen spectra of bioactive substances 01 to 06, respectively, as shown in FIGS. 2 to 7. Then, the chemical structure and chemical name of each bioactive substance were determined from the obtained hydrogen spectrum, as shown in table three below. The code number of the biologically active substance separated and purified, the hydrogen spectrum and the chemical name thereof are related to each other as shown in the third table below.

Watch III

Example three: the total flavone content is determined here, and the relative content of total flavone is expressed in terms of rutin equivalent. First, 0. mu.g/mL, 400. mu.g/mL, 600. mu.g/mL, 1000. mu.g/mL and 1200. mu.g/mL of a standard solution of rutin were prepared with rutin (purchased from ChromaDex) and water. Then, 200. mu.L of the rutin standard solution is taken out of each test tube and put into another new test tube, and 200. mu.L of 5 wt% sodium citrate aqueous solution is added into the new test tubes, mixed uniformly and then kept stand for reaction for 6 minutes. Next, 200. mu.L of a 10 wt% aqueous aluminum nitrate solution (trade name: Alfa Aesar 12360) was added thereto, and after uniformly mixing, the mixture was allowed to stand for 6 minutes to react, and then 2mL of a 4 wt% aqueous sodium hydroxide solution (trade name: Macron 7708-10) was added thereto and uniformly mixed. Finally, 1.4mL of water was added to the test tube and mixed well to obtain a reaction solution, 200. mu.L of the reaction solution in the test tube was put into a 96-well reaction plate, and absorbance was measured at a wavelength of 500nm using an ELISA (enzyme-linked immunosorbent assay) reader (brand: Thermo Fisher Scientific). And (3) drawing the absorbance values of the standard solution of the rutin with different concentrations according to the same method to prepare a calibration curve.

The leaching solution 1 and the leaching solution 4 obtained in example 1 were diluted 10 times by volume with water, and 200. mu.L of the diluted solution was taken out and put into a test tube. Then, 200. mu.L of a 5 wt% aqueous solution of sodium citrate was added thereto, and the mixture was mixed well and allowed to stand for reaction for 6 minutes. Next, 200. mu.L of a 10 wt% aqueous aluminum nitrate solution was added, the mixture was mixed well and allowed to stand for reaction for 6 minutes, and 2mL of a 4 wt% aqueous sodium hydroxide solution was added and mixed well. And finally, adding 1.4mL of water into the test tube, uniformly mixing to obtain reaction liquid, taking 200 mu L of the reaction liquid in the test tube to a 96-hole reaction plate, and detecting the light absorption value by an ELISA reader at the wavelength of 500 nm.

Then, the light absorption value of the diluted leaching solution 4 is calculated into the total flavone content of the leaching solution 4 before dilution by using a calibration curve. Here, it was calculated that the total flavone content of the extract 4 obtained in example 1 was about 815. mu.g/mL, and that the total flavone content of the extract 1 obtained in example 1 was about 432. mu.g/mL.

Example four: cell assay-liver cell antioxidant

The experiment will be divided into four groups of experiment group 1 (the leaching solution 1 obtained in the first addition example and the group treated with hydrogen peroxide), experiment group 2 (the leaching solution 4 obtained in the first addition example and the group treated with hydrogen peroxide), control group (the leaching solution is not added, and the group treated with hydrogen peroxide is not added), and control group (the leaching solution is not added, and the group treated with hydrogen peroxide is not added).

The experimental steps are as follows:

1. human hepatoma cells HepG2 (hereinafter referred to as HepG2 cells) (purchased from American type culture Collection ATCC; Cat. HB-8065) were seeded at 2X 105 cells per well in 6-well plates containing 2ml of medium per well.

Wherein, the cell culture medium: DMEM (from GIBCO, Cat.11965-092) containing 10 vol% Fetal Bovine Serum (FBS) (from Gibco, Cat.10437-028) and 1 vol% penicillin/streptomycin (from Gibco, Cat.15140122).

2. The above culture dish was incubated at 37 ℃ for 24 hours.

3. The cell culture medium in each well of the culture tray was removed.

4. The experimental medium was added to each group and the cells of each group were reacted at 37 ℃ for 1 hour. The experimental media for each group were as follows:

experimental group 1: 2mL of cell culture medium containing 1mg/mL of extract 1 was added to each well.

Experimental group 2: 2mL of cell culture medium containing 1mg/mL of the extract 4 was added to each well.

Control group: 2mL of cell culture medium (without leach solution 4) was added to each well.

Control group: 2mL of cell culture medium (without any leaching solution) was added to each well.

5. In each group, 2. mu.L of cell culture medium containing DCFH-DA solution at a concentration of 5. mu.g/ml was added to each well, and the cells were treated with DCFH-DA for 15 minutes. After treatment, 1mM hydrogen peroxide (H2O2) (purchased from Sigma) was added to the experimental and control groups, and then each group was further incubated at 37 ℃ for 1 hour.

Wherein, 5. mu.g/ml DCFH-DA solution was prepared by dissolving dichlorodihydrofluorescein diacetate (2, 7-dichoro-dihydo-fluorescein diacetate, DCFH-DA; purchased from Sigma; Cat. SI-D6883-50MG) in dimethyl sulfoxide (DMSO; purchased from Sigma, Cat. D2650).

6. Each group was rinsed 1 time with 1mL of 1XPBS (phosphate buffered saline) (available from Gibco, Cat.14200-075) solution per well.

7. Add 200. mu.L of trypsin (purchased from Sigma; Cat.59427C) to each well and react for 5 minutes in the dark, and add 0.6mL of cell culture medium to each well after reaction.

8. The cells and cell culture medium from each well of each group were individually collected into a corresponding 15mL centrifuge tube, and the centrifuge tube containing the cells and cell culture medium was centrifuged at 400xg for 5 minutes.

9. After centrifugation of each group, the supernatant was removed from each group and the cell pellet was redissolved in PBS solution as a cell suspension. The cell suspensions of each group were centrifuged again at 400Xg for 10 minutes.

10. After centrifugation of each group, the supernatant was removed again from each group, and the cell pellet was redissolved in 1XPBS solution as the cell fluid to be tested.

11. The fluorescence signal of DCFH-DA was detected in the cell fluid to be tested in each well using a flow cytometer (brand Beckman; from BD Accuri). The excitation wavelength for fluorescence detection is 450nm-490nm, and the emission wavelength is 510nm-550 nm. Because DCFH-DA is hydrolyzed into DCFH (dichlorodihydrofluorescein) and then oxidized into DCF (dichlorofluorescein) capable of emitting green fluorescence by the active oxygen substance after entering the cells, the fluorescence intensity of the cells treated by DCFH-DA can reflect the content of the active oxygen substance in the cells, and thus the proportion of the number of the cells highly expressed by the active oxygen substance in the cells to the original number of the cells can be obtained. Since the experiment was conducted in duplicate, the measurement results of duplicate experiments of each group were averaged to obtain an average value, and then the average values of the control group and the experimental group were converted into relative ROS production amounts by taking the average value of the control group as 100%, as shown in fig. 8.

The experimental results are as follows:

referring to fig. 8, it can be seen from the results of the control group and the control group that the proportion of cells with high ROS expression (high fluorescence expression) in the control group is greatly increased after the hydrogen peroxide treatment, which indicates that the blue light irradiation indeed causes the generation of reactive oxygen species in the cells, thereby causing the subsequent damage to the human liver cancer cells (HepG2 cells).

The control group generated approximately 12.54 times of the control group, the experimental group 1 generated approximately 4.41 times of the control group, and the experimental group 2 generated approximately 4.37 times of the control group. Therefore, after the cells are treated by the leaching solution 1 and the leaching solution 4, compared with the control group, the experimental group can obviously reduce the generation of ROS. That is, the buckwheat sprout leaching solution can effectively reduce the generation or accumulation of active oxygen substances in cells, can be used as an active oxygen substance scavenger, and can reduce the content of active oxygen substances in cells to reduce the oxidative damage of the cells, wherein the effect of the leaching solution 4 is particularly the best.

Example five: cell assay-detection of GSH content

The experiment will be divided into experiment group 1 (adding the leaching solution 1 obtained in the first example), experiment group 2 (adding the leaching solution 4 obtained in the first example) and control group (not adding any buckwheat seedling leaching solution).

The experimental steps are as follows:

1. human hepatoma cells HepG2 (hereinafter referred to as HepG2 cells) (purchased from American type culture Collection ATCC; Cat. HB-8065) were seeded at 2X 105 cells per well in 6-well culture plates containing 2mL of cell culture medium per well.

Wherein, the cell culture medium: minimal medium (MEM, available from Gibco) was supplemented with additional components to contain 10 vol% fetal bovine serum (FBS, available from Gibco; Cat.10437-028), 1mM sodium pyruvate (sodium pyruvate, available from Gibco), 1.5g/L sodium bicarbonate (available from Gibco), and 0.1mM non-essential amino acids (available from Gibco).

2. Each group was cultured at 37 ℃ for 24 hours. Wherein, the treatment before the culture of each group is as follows:

experimental group 1: 2mg/ml of leach liquor 1 was added to each well.

Experimental group 2: 2mg/ml of the extract 4 was added to each well.

Control group: no leach liquor was added.

3. Cells were collected from each well of each group. Specifically, each group was rinsed 1 time with 1mL of 1XPBS (phosphate buffered saline) (purchased from Gibco; Cat.14200-075) solution, 200. mu.L of trypsin (purchased from Sigma; Cat.59427C) was added to each well and reacted in the dark for 5 minutes, and 0.6mL of cell culture medium was added to each well after the reaction, and the cells and cell culture medium of each well of each group were individually collected into corresponding centrifuge tubes.

4. Each group was rinsed 1 time with PBS solution. Specifically, each centrifugation tube was centrifuged at 400Xg for 5 minutes, the supernatant was removed after centrifugation, a PBS solution was added to the tube for redissolving, and then centrifuged at 400Xg for 5 minutes, and the supernatant was removed after centrifugation to obtain a cell pellet.

5. The cell pellet was reconstituted with PBS solution for each group as a cell suspension.

6. GSH detection stain (purchased from abcam, model Ab112132) was purchased and diluted 1000-fold in volume to obtain GSH detection solution, and each group was stained with GSH detection solution for 15 minutes.

7. Each group was rinsed 1 time with PBS solution. Specifically, each group was centrifuged at 400Xg for 5 minutes, the supernatant was removed after centrifugation, a PBS solution was added to the group to be redissolved, and then centrifuged at 400Xg for 5 minutes, and the supernatant was removed after centrifugation to obtain a cell pellet.

8. The cell sediment is redissolved in 200. mu.L PBS in each group as the cell fluid to be tested.

9. The fluorescence signal of the non-fluorescent Green Dye (non-fluorescent Green Dye) in the test cell fluid of each group was detected using a flow cytometer (Beckman; BD Accuri). The excitation wavelength for fluorescence detection was 490nm and the emission wavelength was 520 nm.

The experimental results are as follows:

referring to fig. 9, if the control group is regarded as 100% Glutathione (GSH) production, the glutathione production of the control group is converted from the glutathione production of the test group 1 to the test group 2 shown in fig. 9, and it can be seen that the glutathione production of the test group 1 is 34% higher than that of the control group, and the glutathione production of the test group 2 is 93% higher than that of the control group. That is, the buckwheat sprout leaching liquor of 0 day and 9 days can promote the production of glutathione in liver cells, wherein the effect of promoting the production of glutathione by the buckwheat sprout leaching liquor of 9 days is better than that of the buckwheat sprout leaching liquor of 0 day.

Glutathione is polypeptide composed of glutamic acid, cysteine and glycine, and its thiol group (G-SH) is easy to combine with free radical, and has antioxidant function. As can be seen from the fifth example, the buckwheat sprout extract for 9 days can improve the generation amount of glutathione in liver cells, and has good antioxidant effect.

Example six: reduction force test

The main principle of the reduction force measurement is that hematite [ K3Fe (CN)6] is reduced into xanthate [ K4Fe (CN)6] (shown as the following formula 7), the prussian blue (shown as the following formula 8) is formed by using Fe3+ in the xanthate, the reduction force is detected through the change of the light absorption value at 700nm, and the higher the light absorption value is, the stronger the reduction force is.

K3Fe (CN)6+ sample → K4Fe (CN)6+ sample-oxide formula 7

Fe3+ + K4Fe (CN)6 → Fe4[ Fe (CN)6]3 (Prussian blue) formula 8

Preparing a solution:

preparing a phosphate buffer solution: 1.34g of anhydrous sodium dihydrogen phosphate (NaH2PO4) (trade name: J.T. Baker 3828-01) and 1.26g of disodium hydrogen phosphate (Na2HPO4) (trade name: Sigma 04270) were weighed out and placed in a measuring flask, and then dissolved in pure water (H2O) and quantified to 100 mL.

Preparing 1 vol% of hematite solution: 1g of hematite (K3Fe (CN)) was weighed out and placed in a quantitative flask, and then dissolved in pure water (H2O) and quantified to 100 mL. (it is stored in the dark, two weeks for storage and at 4 ℃ in an environment).

Preparing 10 vol% Trichloroacetic acid (TCA) solution: 10g of trichloroacetic acid (CCl3COOH) was weighed out and placed in a quantitative flask, and then dissolved in purified water (H2O) and quantified to 100 mL.

Preparing 0.1 vol% ferric chloride (FeCl3) solution: 0.1g of ferric chloride was weighed out and placed in a quantitative flask, and then dissolved in pure water (H2O) and quantified to 100 mL. (it is required to be stored in a dark place, in a daily environment at 4 ℃ C.)

Preparing 1mg/mL vitamin C (Vit C) solution: 10mg of L-Ascorbic acid (L-Ascorbic acid) was weighed out and placed in a quantitative flask, and then dissolved in purified water (H2O) and quantified to 10 mL.

(need to be configured on the same day)

The experimental steps are as follows:

1mL of a 1mg/mL vitamin C solution was taken and placed in a quantitative flask, and then water (H2O) was added and quantified to 10mL to obtain 100. mu.g/mL vitamin C solution.

The vitamin C solution 100. mu.g/mL was used to prepare standard solutions of 0. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 60. mu.g/mL, 80. mu.g/mL and 100. mu.g/mL in glass tubes, respectively, in the manner shown in Table four.

Watch four

Then, 250. mu.L of each concentration of standard solution (i.e., specimen) was placed in different tubes, and 250. mu.L of phosphate buffer solution was added to each tube, and the solutions were uniformly mixed by vortexing (vortex). Then, 250. mu.L of a hematite solution (1 vol%) was added to each tube, and the solution was uniformly mixed by vortexing (vortex). Next, each tube was placed in a water bath at 50 ℃ for 20 minutes. Then, 250. mu.L of trichloroacetic acid solution (10 vol%) was added to each tube, and the solution was uniformly mixed by vortexing (vortex). Next, the mixture was centrifuged at 3000g for 10 minutes. Then, 30. mu.L of the supernatant was taken from each tube, 300. mu.L of pure water was added, and 120. mu.L of ferric chloride solution (0.1 vol%) was further added, and the solution was uniformly mixed by vortexing (vortex) and reacted for 10 minutes to obtain a reaction solution. mu.L of the reaction solution was transferred to a 96-well reaction plate, and absorbance was measured at a wavelength of 700nm using an ELISA reader (brand: Thermo Fisher Scientific). The absorbance values of six vitamin C solutions with different concentrations, which were measured in the same manner, were plotted as calibration lines.

250 μ L each of the leachate 1 (i.e., sample) obtained in example one and the leachate 4 (i.e., sample) obtained in example one was taken out of each test tube, and 250 μ L of phosphate buffer solution was added to each test tube, and the solutions were uniformly mixed by vortexing (vortex). Next, 250. mu.L of a hematite solution (1 vol%) was added to each tube, and the solution was uniformly mixed by vortexing (vortex). Next, each tube was placed in a water bath at 50 ℃ for 20 minutes. Next, 250. mu.L of trichloroacetic acid solution (10 vol%) was added to each tube, and the solution was uniformly mixed by vortexing (vortex). Next, each tube was centrifuged at 3000g for 10 minutes. Then, 30. mu.L of the supernatant was taken from each tube, 300. mu.L of water was added, and 120. mu.L of ferric chloride solution (0.1 vol%) was further added, and the solution was uniformly mixed by vortex (vortex) and reacted for 10 minutes to obtain a reaction solution. 200. mu.L of the reaction solution in the test tube was taken into a 96-well reaction plate, and absorbance was measured at a wavelength of 700nm with an ELISA reader.

The experimental results are as follows:

referring to fig. 10, the absorbance values of the diluted 9-day buckwheat sprout leachate and 0-day buckwheat sprout leachate are converted into the reducing power corresponding to the vitamin C content by using a calibration curve. It can be seen that the reducing power of the buckwheat sprout leaching solution of day 0 before dilution (i.e., leaching solution 1 obtained in example 1) corresponds to the reducing power of vitamin C (L-Ascorbic Acid Sodium Salt) having a content of about 437 μ g/mL, and the reducing power of the buckwheat sprout leaching solution of day 9 before dilution (i.e., leaching solution 4 obtained in example 1) corresponds to the reducing power of vitamin C having a content of about 1752 μ g/mL, so that it is assumed that the reducing power of leaching solution 4 is significantly higher than that of leaching solution 1, i.e., the buckwheat sprout leaching solution obtained by using the buckwheat sprout of day 9 as a raw material has a stronger reducing power than that of the non-germinated seed.

Example seven: cell assay-detection of GSH content

Here, the cell test was conducted using the separation liquid collected from the leaching solution 4 at time point A, C, D, E, F and G in the second experiment (I) and after removing the solvent, six bioactive substances (as shown in Table five below) were obtained.

Watch five

	Point in time	Name of ingredient
			Biologically active substance 01	A	Guanosine
Biologically active substance 02	E	Vitexin
			Biologically active substance 03	G	Isovitexin
Biologically active substance 04	F	Rutin (Cycleic acid)
			Biologically active substance 05	D	Oriental elements
Biologically active substance 06	C	All-grass of common Melilotus8-C-glucoside

The experiment will be divided into experiment group 1 (adding bioactive substance 01), experiment group 2 (adding bioactive substance 02), experiment group 3 (adding bioactive substance 03), experiment group 4 (adding bioactive substance 04), experiment group 5 (adding bioactive substance 05), experiment group 6 (adding bioactive substance 06) and control group (not adding bioactive substance).

The experimental steps are as follows:

experimental group 1: 2mg/ml of biologically active substance 01 are added per well.

Experimental group 2: 2mg/ml of biologically active substance 02 was added to each well.

Experimental group 3: 2mg/ml of biologically active substance 03 are added per well.

Experimental group 4: the biologically active substance 04 is added to each well at a concentration of 2 mg/ml.

Experimental group 5: 2mg/ml of biologically active substance 05 was added per well.

Experimental group 6: 2mg/ml of biologically active substance 06 per well.

Control group: no biologically active substance, i.e. only 2mL of cell culture medium alone, was added to each well.

4. Each group was rinsed 1 time with PBS solution. Specifically, each group was centrifuged at 400Xg for 5 minutes, the supernatant was removed after centrifugation, a PBS solution was added to the group to be redissolved, and then centrifuged at 400Xg for 5 minutes, and the supernatant was removed after centrifugation to obtain a cell pellet.

The experimental results are as follows:

referring to FIG. 11, in the HepG2 cell treated with the biologically active substance 01 (guanosine), the glutathione production amount was 1.6 times that of the control group; the production amount of Glutathione (GSH) of HepG2 cell treated by the bioactive substance 02 (vitexin) is 0.6 times of that of the control group; the generation amount of glutathione of HepG2 cells treated by the bioactive substance 03 (isovitexin) is 0.4 times that of the control group; the generation amount of glutathione of HepG2 cells treated by the bioactive substance 04 (rutin) is 0.6 times of that of the control group; the yield of glutathione in the liver cells treated by the bioactive substance 05 (isoorientasin) is 0.6 times that of the liver cells treated by the control group; the yield of glutathione in HepG2 cells treated with the bioactive substance 06 (luteolin 8-C-glucoside) was 0.4 times that in the control group.

Glutathione is polypeptide composed of glutamic acid, cysteine and glycine, and its thiol group (G-SH) is easy to combine with free radical, and has antioxidant function. As can be seen from fig. 11, the bioactive substance 01 separated from the leaching solution 4 can increase the amount of glutathione produced in the liver cells, and thus has a good antioxidant effect.

In summary, in any embodiment, the use of buckwheat sprout leachate for preparing a composition for enhancing cellular antioxidant and/or liver health and a method for preparing buckwheat sprout leachate are provided, which are suitable for providing buckwheat sprout leachate or a composition thereof. Wherein the buckwheat sprout leaching liquor or the composition thereof has one or more of the following functions: improving the antioxidant activity of cells, improving the metabolic activity, and improving the synthesis of Glutathione (GSH) in cells.

Claims

1. Use of buckwheat seedling leaching solution in preparing composition for improving cell antioxidant activity is provided.

2. The application of buckwheat seedling leaching liquor in preparing liver health composition.

3. The use of the buckwheat sprout leachate of claim 2, wherein the buckwheat sprout leachate is used to increase the antioxidant and metabolic activity of the cells.

4. The use of the buckwheat sprout leachate of claim 3, wherein the buckwheat sprout leachate is used to enhance the synthesis of Glutathione (GSH) in cells.

5. The use of the buckwheat sprout leachate of claim 1 or 2, wherein the buckwheat sprout leachate is obtained by leaching buckwheat sprouts, wherein the buckwheat sprouts are seedlings of buckwheat seeds that have been germinated and grown for 6 to 12 days.

6. The use of the buckwheat sprout extract of claim 5, wherein the solvent is water, the weight ratio of the solvent to the buckwheat sprout is 2:1 to 5:1, the extraction temperature of the buckwheat sprout is between 80 ℃ and 90 ℃, and the extraction time of the buckwheat sprout is 120 minutes.

7. The use of buckwheat sprout leachate solution of claim 6, wherein the buckwheat sprout leachate solution is 4.7 ° Bx to 5.3 ° Bx.

8. The use of the buckwheat sprout leachate of claim 6, wherein the flavone content of the buckwheat sprout leachate is about 815 μ g/mL.

9. Use of buckwheat sprout leachate according to claim 1 or 2, wherein the buckwheat sprout leachate comprises at least one of the following bioactive substances: guanosine (Guanosine), Vitexin (Vitexin), Isovitexin (Isovitexin), Rutin (Rutin), isoorientaxin (Isoorientin), and Luteolin 8-C-glucoside (Luteolin-8-C-glucoside).

10. The use of the buckwheat sprout leachate of claim 9, wherein the guanosine is used to enhance the synthesis of Glutathione (GSH) in the cells.

11. A method for preparing buckwheat seedling leaching liquor comprises:

leaching water and buckwheat seedlings at a temperature of 80-90 ℃ for 60-180 minutes to obtain a leaching stock solution, wherein the weight ratio of the water to the buckwheat seedlings is 2: 1-5: 1; and

filtering the raw extract to obtain a buckwheat sprout extract.

12. The method of claim 11, further comprising:

concentrating the buckwheat sprout leaching liquor under reduced pressure until the buckwheat sprout leaching liquor is 4.7 ° Bx to 5.3 ° Bx to obtain the concentrated buckwheat sprout leaching liquor.

13. The method of claim 11, wherein the vacuum concentration step is performed at 50 ℃ to 60 ℃.

14. The method of claim 11, wherein the buckwheat sprout leachate after concentration has a total flavone content of 815 μ g/mL.

15. The method according to claim 11, wherein the buckwheat seedlings are seedlings of buckwheat seeds that grow for 6 to 12 days after germination.

16. The method of claim 11, wherein the buckwheat sprout leachate comprises at least one of the following bioactive substances: guanosine (Guanosine), Vitexin (Vitexin), Isovitexin (Isovitexin), Rutin (Rutin), isoorientaxin (Isoorientin), and Luteolin 8-C-glucoside (Luteolin-8-C-glucoside).

17. The method of claim 11, wherein the guanosine is used to enhance Glutathione (GSH) synthesis in the cell.

18. Buckwheat sprout leachate, comprising at least one of the following bioactive substances: guanosine (Guanosine), Vitexin (Vitexin), Isovitexin (Isovitexin), Rutin (Rutin), isoorientaxin (Isoorientin), and Luteolin 8-C-glucoside (Luteolin-8-C-glucoside).